We investigate skin optical clearing in laboratory animals ex vivo and in vivo by means of low-molecular-weight paramagnetic contrast agents used in magnetic resonance imaging (MRI) and a radiopaque agent used in computed tomography (CT) to increase the sounding depth and image contrast in the methods of fluorescence laser imaging and optical coherence tomography (OCT). The diffusion coefficients of the MRI agents Gadovist®, Magnevist®, and Dotarem®, which are widely used in medicine, and the Visipaque® CT agent in ex vivo mouse skin, are determined from the collimated transmission spectra. MRI agents Gadovist® and Magnevist® provide the greatest optical clearing (optical transmission) of the skin, which allowed: 1) an almost 19-fold increase in transmission at 540 nm and a 7 – 8-fold increase in transmission in the NIR region from 750 to 900 nm; 2) a noticeable improvement in OCT images of skin architecture; and 3) a 5-fold increase in the ratio of fluorescence intensity to background using TagRFP-red fluorescent marker protein expressed in a tumour, after application to the skin of animals in vivo for 15 min. The obtained results are important for multimodal imaging of tumours, namely, when combining laser fluorescence and OCT methods with MRI and CT, since the contrast agents under study can simultaneously enhance the contrast of several imaging methods.

The results of numerical simulation of the near-field distribution inside and in the vicinity of two types of gold nanoparticles (nanospheres and nanorods) intended for producing complexes of gold nanoparticles linked with proteins and exciting photosensitizers in the wavelength range of 532 – 770 nm are presented. Quantitative estimates of the field localisation (enhancement) are obtained depending on the type of gold nanoparticles and dimensional factors. The tendency of the red shift of the wavelength at which the maximum local field enhancement is achieved relative to the positions of the maxima of the absorption and scattering cross sections of nanoparticles and complexes is described.

This study reports the development of a binary drug delivery system consisting of charged liposomes and an oppositely charged peptide-photosensitiser conjugate. Liposomes were prepared with phosphatidyl-l-serine as a negatively charged lipid. Calcein, a fluorophore marker, and doxorubicin, an anticancer drug, were used as model hydrophilic loads. The conjugate consisted of a positively charged arginine-rich peptide synthesised by solid-phase peptide synthesis, and a phthalocyanine derivative with characteristic absorption around 685 nm. Illumination of the binary system with far-red light of 12-15 mW/cm(2) intensity resulted in 5- to 15-fold increase in release of payloads from the liposomes. The mechanism of drug release was based on photosensitised oxidation of lipids destabilising the liposomal membrane. The cytotoxicity of the liposomes loaded with doxorubicin was tested on B16-F10 melanoma and Y79 retinoblastoma cells. The cytotoxicity of the illuminated binary system in melanoma cell line was significantly higher as compared to the system without illumination. The components of the binary system can be individually prepared and stored with greater storage stability. However, their combination will allow for substantial release of hydrophilic payload from the liposomes under externally applied light.

An EGFR-targeting peptide with Ala-Glu-Tyr-Leu-Arg sequence was assembled directly onto the surface of spray-dried chitosan microparticles using solid-phase peptide synthesis with Fmoc chemistry. Both targeted and scrambled peptides were cleaved from chitosan with enzymatic digestion, isolated using reversed-phase HPLC, then identified with LC/MS/MS and amino acid analysis. Particles with conjugated peptides and fluorescent-labeled were characterized for binding with A549 (cancer) and WI-26 VA4 (normal) lung cells using flow cytometry and confocal microscopy. The purity of peptide synthesis was in the range of 74–77%. The cell binding studies revealed that particles modified with the peptide bind to lung cancer cells 8.3 times higher than the normal lung cells. The binding potential of surface-modified targeted particles to the tumor cells was compared with scrambled and unmodified ones and was found to be significantly different. The binding was 7.3–7.5-fold higher than the scrambled and unmodified particles, respectively. Modification of chitosan particles with direct assembly of Ala-Glu-Tyr-Leu-Arg peptide on their surface enhanced their targeting potential to lung cancer cells and could be used as a potential carrier for delivery and therapy. This approach can further be utilized for assembly of other short peptide ligands on micro- or nanoparticulate systems for tumor targeting.

The present work is devoted to the study of pharmacokinetics of infrared photosensitizer (PS) based on hydroxyaluminium tetra‑3‑phenylthiophthalocyanine in a sterically stabilized liposomal form. The study was carried out on adult female mice. The PS was administered once intravenously at a dose of 6 mg / kg. Evaluation of the PS accumulation dynamics in the mice tissues and organs was performed at time intervals from 5 minutes to 7 days using spectral‑fluorescent method. The maximum accumulation of the PS photoactive form was recorded in lungs (32 µg / g in the interval of 5–30 minutes after introduction), liver (20.8 µg / g in the interval of 4–24 hours after introduction) and spleen (28 µg / g 4 hours after introduction). At the same time, by the end of the observation period (7 days after administration), trace amounts of the PS photoactive form were still detected in the liver and the spleen at a calculated concentration of 0.5‑1 µg / g. The PS accumulated the least in muscles and skin. The fluorescent signal from the PS accumulated in skin was detectable almost immediately, and its concentration remained at the same level (1.2‑1.5 µg / g) for up to 3 days of observation. In the muscles, the concentration of the PS reached 1.5 µg / g 15 minutes after administration, and then gradually decreased until 0.25 µg / g at 24 hours. Data on the pharmacokinetics of PS in blood, basic organs and tissues of animals were obtained, pharmacokinetic parameters were calculated. 7 days after the administration, the PS concentration in the skin and muscles was below the detection limit. The studies confrmed that PEGylation of the PS liposomal form slows down the process of its capture by reticulo‑endothelial system. It was shown that the PS circulates in blood and organs of mice for a long time and it completely distributes only when 4 hours pass after administration.

The present work is devoted to the study of pharmacokinetics of infrared photosensitizer (PS) based on hydroxyaluminium tetra‑3‑phenylthiophthalocyanine in a sterically stabilized liposomal form. The study was carried out on adult female mice. The PS was administered once intravenously at a dose of 6 mg / kg. Evaluation of the PS accumulation dynamics in the mice tissues and organs was performed at time intervals from 5 minutes to 7 days using spectral‑fluorescent method. The maximum accumulation of the PS photoactive form was recorded in lungs (32 µg / g in the interval of 5–30 minutes after introduction), liver (20.8 µg / g in the interval of 4–24 hours after introduction) and spleen (28 µg / g 4 hours after introduction). At the same time, by the end of the observation period (7 days after administration), trace amounts of the PS photoactive form were still detected in the liver and the spleen at a calculated concentration of 0.5‑1 µg / g. The PS accumulated the least in muscles and skin. The fluorescent signal from the PS accumulated in skin was detectable almost immediately, and its concentration remained at the same level (1.2‑1.5 µg / g) for up to 3 days of observation. In the muscles, the concentration of the PS reached 1.5 µg / g 15 minutes after administration, and then gradually decreased until 0.25 µg / g at 24 hours. Data on the pharmacokinetics of PS in blood, basic organs and tissues of animals were obtained, pharmacokinetic parameters were calculated. 7 days after the administration, the PS concentration in the skin and muscles was below the detection limit. The studies confrmed that PEGylation of the PS liposomal form slows down the process of its capture by reticulo‑endothelial system. It was shown that the PS circulates in blood and organs of mice for a long time and it completely distributes only when 4 hours pass after administration.

Light-induced drug release has been explored as a strategy for externally modulating the release of drug from delivery systems. This study reports the development of a solid lipid nanoparticulate system (SLN) for paclitaxel (PTX), where photosensitizer-mediated oxidation of lipids was used as a mechanism for controlling the drug release. Low-intensity (23 mW/cm(2)) near-infrared (around 730 nm) illumination was externally applied as the light source. PTX release was less than 10% within 4 h from these SLN and was 8-fold higher after application of light at time zero. The other advantages of this approach include the use of ascorbic acid (ASC) as an antioxidant for enhancing the release and storage stability of the delivery system. Antioxidant like ASC in the SLN decrease the degradation of lipid by 8-fold within 4 months of storage. Presence of ASC and light illumination of SLN containing PTX further decreased the IC50 by 2 times in A549 cells. The uniqueness of this approach allows the possibility of external modulation to achieve pulsatile release from the delivery system. The light used in the NIR spectral range of 700-850 nm, which has the greatest tissue penetration ability, with a low intensity will be safe for normal tissues.

Over the last several decades, nanoparticulate delivery systems have emerged as advanced drug and gene delivery tools for cancer therapy. However, their translation into clinical use still poses major challenges. Even though many innovative nanoparticulate approaches have shown very positive results both in vitro and in vivo, few of them have found a place in clinical practice. Possible factors responsible for the existing gap in the translation of nanomedicine to clinical practice may include oversimplification of enhanced permeability and retention effect, lack of correlation between the in vivo animal data vs their translation in human, and challenging multiple biological steps experienced during systemic delivery of nanomedicine. Understanding these challenges and coming up with solutions to overcome them is an important step in effective translation of nanomedicine into clinical practice. This review focuses on advancements in the field of nanomedicine used for anti-cancer therapy, including passive targeting, active targeting, and stimuli-controlled delivery. The review further reveals some of the challenges that are currently faced by pharmaceutical scientists in translation of nanomedicine; these include lack of adequate models for preclinical testing that can predict efficacy in humans, absence of appropriate regulatory guidelines for their approval processes, and difficulty in scale-up of the manufacturing of nanodrug delivery systems. A better understanding of these challenges will help us in filling the gap between the bench and bedside in cancer therapy.

Background. Current work is devoted to the study in vivo of concentration and selectivity of accumulation of infrared photosensitizer (PS) hydroxyaluminium tetra-3-phenylthiophthalocyanine in liposomal form in intramuscularly and subcutaneously transplanted mice tumor models in comparison to normal tissues. Objective: to study the level and selectivity of accumulation of hydroxyaluminium tetra-3-phenylthiophthalocyanine liposomal form on mice tumor models in order to optimize the transplantation approach and the starting of photodynamic treatment. Materials and methods. A range of transplantable mice tumors was used in the study: solid carcinoma Ehrlich (ELD) and solid sarcoma S-37, epidermoid Lewis lung carcinoma (LLC) and colon adenocarcinoma (AKATOL). For the assessment of concentration of PS in tissues was evaluated by fluorescence spectroscopy in vivo. Results. The optimum transplantation approaches were shown to be as follows. Solid carcinoma Ehrlich (ELD) provided the highest accumulation of PS when transplanted intramuscularly. Five hours after administration concentration of PS in tumor achieves more than 7 mg/kg, with selectivity in comparison to normal tissue 3 : 1. The maximum concentration of PS in sarcoma S-37 was observed with subcutaneous transplantation, achieving at 5 h after administration the value of 5.4 mg/kg with selectivity of accumulation 4.3: 1. Both LLC and AKATOL showed optimum results with intramuscular transplantation. Maximum concentration of PS in LLC was observed 5 h after administration, achieving 7.5 mg/kg with selectivity exceeding 4. Concentration of photosensitizer in AKATOL 7h post administration achieved 6.8 mg/kg with selectivity about 2. Conclusions. Liposomal form of PS with intravenous administration selectively accumulates in tumors. The obtained experimental data allows to recommend the method of listed tumors models transplantation for the studies of PS.

This work is devoted to the study of two new synthetic bacteriochlorins with four and eight cationic substitutes as the photosensitizers in the photodynamic process. The spectral and antibacterial properties of these photosensitizers in saline solution were investigated. It is shown, that the aggregation ability decreases and the antibacterial efficiency grows as the cationic substitute number increases.

The research is devoted to development and study of photosensitizers on a base of injectable nanoparticular formulations of phthalocyanine derivatives absorbing at long wavelength (near-infrared) range of 710–740 nm: Thiosens [liposomal form of aluminum hydroxide 1,8,15,22-tetrakis(phenylsulfanyl)phthalocyanine] and Octasens [polymeric micellar form of zinc 1,4,8,11,15,18,22,25-octachloro-2,3,9,10,16,17,23,24-octakis-(decylsulfanyl)phthalocyanine based on domestic poloxamer Emuxol-268]. Experiments have shown that the photosensitizers under study show high selectivity of accumulation and prolonged retaining at high concentration in tumor, relatively fast clearance from normal tissue and high therapeutic efficiency of photodynamic treatment on model tumors.

The work is devoted to study of new prototype contrasting agents for magnetic resonance imaging on a base of manganese and gadolinium sulphophthalocyanines. Candidate complexes were shown to possess the R1 molar relaxivity values comparable or exceeding ones for commercially available clinical contrast agents such as Magnevist. Intravenous administration of aqueous solution of studied complexes resulted in significant enhancement of contrast of C6 glioma imaging under T1-weighted protocol, allowing to consider them as perspective for further development. Near infrared photosensitizers based on nanoparticular forms of phthalocyanine derivatives.

The work is devoted to the study of the action of photosensitizer Thiosens on the fluorescent tumor model mel Kor-TurboRFP. It was shown that cell line mel Kor-TurboRFP is sensitive to the phototoxic action of Thiosens in vitro. That allows to use of fluorescent tumor mel Kor-TurboRFP to study the photodynamic action of Thiosens in vivo. Non-invasive monitoring of fluorescence of tumor mel Kor-TurboRFP sensitized by Thiosens allows independent control of the dynamics of the three-dimensional distribution of the fluorescent tumor and the photosensitizer. This makes it possible to optimize the PDT approach, allowing the most optimal way to choose the time to start of tumor irradiation, irradiation time and the irradiated area patterns.

The implementation of liposome delivery systems ensures the intravenous introduction of hydrophobic photosensitizers used in the treatment of tumors by the photodynamic therapy method with their preservation in a therapeutically effective nonaggregated form and high accumulation selectivity in the tumor. Phthalocyanines are one of the fastest growing classes of antineoplastic photosensitizers, most of which are hydrophobic compounds. In this study the authors have developed the unique composition and technology of preparing the lyophilized liposomal formulation of infrared photosensitizer: aluminum hydroxide tetra-3-phenylthiophtalocyanine (Thiosens). At the stage of biological experiments on model mouse tumors, the preservation of high accumulation selectivity in the tumor (selectivity index is higher than 4) and antineoplastic effectiveness (inhibition of tumor growth is from 70 to 94%) were found during the transition from the previously studied model dosage form to the created lyophilized liposomal one. To estimate chemical and pharmaceutical criteria and the photodynamic efficiency of the photosensitizer on a number of transplanted mice tumor models in vivo, we have conducted a preclinical study which has demonstrated the stability of antitumor activity and quality consistency of the developed dosage form for one and a half years.

Authors have developed the composition, technology of preparation. dosage form and sucrose-protected lyophilization protocol for formulation for systemic administration of near-infrared range photosensitizer thiosens based on PEG-liposomal formulation of aluminum hydroxide tetra-3-phenylthiophthalocyanine derivative. Optimization of preparations allowed to formulate the quality control and standardization criteria for the drug "thiosens liposomal lyophilisate for injection 1.5 mg" and establish a pharmacopoeia monograph for the drug candidate. Preclinical studies of chosen pharmachemical criteria and photodynamic efficiency of photosensitizer on a number of transplanted mice tumor models in vivo demonstrated stability of antitumor activity and quality consistency of the photosensitizer over about 15 year what allow to recommend the shelf life at least of 1 year.

Dye family of naphthalocyanines and their relatives are potent photosensitizers for photodynamic therapy of tumors. One of the best photosensitizers in this series is tiosens – the original domestic product, created by FGUP GNC “NIOPIK”. It is hydrophobic aluminum hydroxide tetra-3-phenylthiophthalocyanine absorbing in a infrared spectral range (absorption maximum at 717 nm). With the purpose of targeted delivery tiosens in a tumor and overcoming of insufficient solubility of liposomal form is developed. Experiments revealed the preservation of physical and chemical properties and high photodynamic activity of the liposomal photosensitizer for the duration of storage. References Krasnopol'skij Ju.M., Stepanov A.E., Shvec V.I. / Biofarm. zh. 2009. №3. P.18-29. Mishra B., Patel B.B., Tiwari S. / Nanomed. Nanotechnol., Biol., Med. 2010. V. 6. P. 9–24. Meerovich I.G., Oborotova N.A. / Ros. bioterapevt. zh. 2007. №4. P.3-8. Allison R.R., Sibata C.H. / Photodiag. Photodyn. Ther. 2010. V. 7. P.61-75. Meerovich I.G, Smirnova Z.S., Oborotova N.A. / Bul. Exper. Biol. Med. 2005. V.139. P.427-430.

BACKGROUND:Fluorescently labeled cell-penetrating peptides can translocate into cells by endocytosis and upon light irradiation, lyse the endocytic vesicles. This photo-inducible endosomolytic activity of Fl-CPPs can be used to efficiently deliver macromolecules such as proteins and nucleic acids and other small organic molecules into the cytosol of live cells. The requirement of a light trigger to induce photolysis provides a more spatial and temporal control to the intracellular delivery process. METHODS:In this report, we examine the molecular level mechanisms by which cell-penetrating peptides such as TAT when labeled with small organic fluorophore molecules acquire a photo-induced lytic activity using a simplified model of lipid vesicles. RESULTS:The peptide TAT labeled with 5(6)-carboxytetramethylrhodamine binds to negatively charged phospholipids, thereby bringing the fluorophore in close proximity to the membrane of liposomes. Upon light irradiation, the excited fluorophore produces reactive oxygen species at the lipid bilayer and oxidation of the membrane is achieved. In addition, the fluorescent peptide causes aggregation of photo-oxidized lipids, an activity that requires the presence of arginine residues in the peptide sequence. CONCLUSIONS:These results suggest that the cell-penetrating peptide plays a dual role. On one hand, TAT targets a conjugated fluorophore to membranes. On the other hand, TAT participates directly in the destabilization of photosensitized membranes. Peptide and fluorophore therefore appear to act in synergy to destroy membranes efficiently. GENERAL SIGNIFICANCE:Understanding the mechanism behind Fl-CPP mediated membrane photodamage will help to design optimally photo-endosomolytic compounds.

The optimum composition of a stable lyophilized liposomal formulation of tiosens is established and includes lecithin, cholesterol, and PEG-2000-DSPE in a molar ratio of 1:0.22:0.002 and sucrose solution as a cryoprotector in a 1:9 lecithin:cryoprotector weight ratio. The effects of sonication, homogenization, and filtration on the quality of the liposomal preparation were studied. The results of preliminary studies lead to a conclusion about the satisfactory storage stability of the liposome form of tiosens.